Mass spectrometers (MS) are used widely for identifying and quantifying molecular species in a sample. During analysis, molecules from the sample are ionized to form ions that are introduced into the mass spectrometer for analysis. The mass spectrometer measures the mass-to-charge ratio (m/z) and intensity of the introduced ions.
Mass spectrometers are limited in the number of different ions reliably detected and quantified within a single sample spectrum. As a result, samples containing many molecular species may produce spectra that are too complex for interpretation or analysis using conventional mass spectrometers.
In addition, the concentration of molecular species often varies over a wide range. Biological samples, for example, typically have a greater number of molecular species at lower concentrations than at higher concentrations. Thus, a significant fraction of ions appear at low concentration. The low concentration is often near the detection limit of common mass spectrometers. Moreover, at low concentration, ion detection suffers from background noise and/or interfering background molecules. Consequently, detection of such low abundance species can be improved by removing as much of the background noise as possible and reducing the number of interfering species that are present in the spectrum.
A chromatographic separation, prior to injecting the sample into the mass spectrometer, is commonly used to reduce the complexity of such spectra. For example, peptides or proteins often produce clusters of ions that elute at a common chromatographic retention time and thus produce peaks that overlap in a spectrum. Separating the clusters from the different molecules, in time, helps to simplify interpretation of the spectra produced by such clusters.
Common chromatographic separation instruments include gas chromatographs (GC) and liquid chromatographs (LC). When coupled to a mass spectrometer, the resulting systems are referred to as GC/MS or LC/MS systems. GC/MS or LC/MS systems are typically on-line systems in which the output of the GC or LC is coupled directly to the MS.
A combined LC/MS system provides an analyst with a powerful means to identify and to quantify molecular species in a wide variety of samples. Common samples contain a mixture of a few or thousands of molecular species. The molecules often exhibit a wide range of properties and characteristics, and each molecular species can yield more than one ion. For example, the mass of a peptide depends on the isotopic forms of its nucleus, and an electrospray interface can ionize peptides and proteins into families of charge states.
In an LC/MS system, a sample is injected into the liquid chromatograph at a particular time. The liquid chromatograph causes the sample to elute over time resulting in an eluent that exits the liquid chromatograph. The eluent exiting the liquid chromatograph is continuously introduced into the ionization source of the mass spectrometer. As the separation progresses, the composition of the mass spectrum generated by the MS evolves and reflects the changing composition of the eluent.
Typically, at regularly spaced time intervals, a computer-based system samples and records the spectrum. In conventional systems, the acquired spectra are analyzed after completion of the LC separation.
After acquisition, conventional LC/MS systems generate one-dimensional spectra and chromatograms. The response (or intensity) of an ion is the height or area of the peak as seen in either the spectrum or the chromatogram. To analyze spectra or chromatograms generated by conventional LC/MS systems, peaks in such spectra or chromatograms that correspond to ions must be located or detected. The detected peaks are analyzed to determine properties of the ions giving rise to the peaks. These properties include retention time, mass-to-charge ratio and intensity.
Mass or mass-to-charge ratio (m/z) estimates for an ion are derived through examination of a spectrum that contains the ion. Retention time estimates for an ion are derived by examination of a chromatogram that contains the ion. The time location of a peak apex in a single mass-channel chromatogram provides an ion's retention time. The m/z location of a peak apex in a single spectral scan provides the ion's m/z value.
A conventional technique for detecting ions using an LC/MS system forms a total ion chromatogram (TIC). Typically, this technique is applied if there are relatively few ions requiring detection. A TIC is generated by summing, within each spectral scan, all responses collected over all m/z values and plotting the sums against scan time. Ideally, each peak in a TIC corresponds to a single ion.
Co-elution of peaks from multiple molecules is one possible problem with this method of detecting peaks in a TIC. As a result of co-elution, each isolated peak seen in the TIC may not correspond to a unique ion. A conventional method for isolating such co-eluted peaks is to select the apex of one peak from the TIC and collect spectra for the time corresponding to the selected peak's apex. The resulting spectral plot is a series of mass peaks, each presumably corresponding to a single ion eluting at a common retention time.
For complex mixtures, co-elution also typically limits summing of spectral responses to sums only over a subset of collected channels, e.g., by summing over a restricted range of m/z channels. The summed chromatogram provides information about ions detected within the restricted m/z range. In addition, spectra can be obtained for each chromatographic peak apex. To identify all ions in this manner, multiple summed chromatograms are generally required.
Another difficulty encountered with peak detection is detector noise. A common technique for mitigating detector noise effects is to signal-average spectra or chromatograms. For example, the spectra corresponding to a particular chromatographic peak can be co-added to reduce noise effects. Mass-to-charge ratio values as well as peak areas and heights can be obtained from analyzing the peaks in the averaged spectrum. Similarly, co-adding chromatograms centered on the apex of a spectral peak can mitigate noise effects in chromatograms and provide more accurate estimates of retention time as well as chromatographic peak areas and heights.
Aside from these problems, additional difficulties are encountered when conventional peak detection routines are used to detect chromatographic or spectral peaks. If performed manually, such conventional methods are both subjective and tedious. When performed automatically, such methods can still be subjective, due to a subjective selection of thresholds for identification of peaks. Further, these conventional methods tend to be inaccurate because they analyze data using only a single extracted spectrum or chromatogram, and do not provide ion parameter estimates having the highest statistical precision or lowest statistical variance. Finally, conventional peak-detection techniques do not necessarily provide uniform, reproducible results for ions at low concentration, or for complex chromatograms, where co-elution and ion interference tend to be common problems.